Unlike higher animals, folate biosynthesis is an essential bacterial pathway, and it is an attractive target for the development of new antibacterial agents. The sulfa drugs target a key enzyme in the pathway, dihydropteroate synthase (DHPS), but bacterial resistance is making them less potent in clinical therapy. Sulfa drugs mimic the binding of p-arninobenzoic acid (pABA) to DHPS, but the second pterin binding site has yet to be developed in the search for novel inhibitors. This site is buried in the protein core, and mutations that can lead to resistance are less likely to be structurally tolerated than those in the pABA binding site, which comprises flexible loops. A barrier to developing new DHPS inhibitors is our incomplete understanding of the catalytic mechanism. Four structures of the enzyme are available, but the loop regions have not been visualized in their functional conformation. In contrast, the rigid and conserved pterin-binding site has been clearly defined. We propose to design and synthesize transition state analogues of DHPS and other inhibitors that access the pterin binding site, and to iteratively use them to characterize the catalytic mechanism and to generate inhibitor scaffolds for a future drug discovery program. This project is being submitted in the high risk, high impact category under PA-03-080 (biodefense and emerging infectious disease) for three reasons. First, there is no guarantee that useful small molecules will be forthcoming from the project, but the discovery of the exact catalytic mechanism of this enzyme and tight pterin binding scaffolds would be a major advance in pursuing this well validated drug target. Second, we have solved the structure of DHPS from the category A biowarfare agent B. anthracis, and this enzyme, together with those from Y, pestis and F. tularensis will be the focus of this application. Our goal is to design related inhibitors with activity spectra tuned to these three enzymes that could form the basis of biowarfare therapeutics. Third, the pterin-binding pocket of DHPS offers the opportunity of also developing desperately needed broad-spectrum anti-infectives. The project will be a collaboration between a crystallographer at St. Jude Children's Research Hospital and a medicinal chemist at the University of Tennessee Health Science Center. The institutions have complementary programs in infectious diseases with a strong emphasis on studying bioterrorism.